ORIGINAL PAPER

International Journal of Occupational Medicine and Environmental Health 2018;31(4):1 – 13

https://doi.org/10.13075/ijomeh.1896.01037

SUBCUTANEOUS ADMINISTRATION
OF INFLIXIMAB-ATTENUATED
SILICA-INDUCED LUNG FIBROSIS
HUA ZHANG1,2, JUN-NA SUI3, LEI GAO4, and JIAN GUO3
1
Qilu Hospital of Shandong University, Jinan, China
Department of Respiratory Medicine
2
Qingdao Central Hospital, Qingdao, China
Department of Occupational Disease
3
Qingdao Central Hospital, Qingdao, China
Department of Clinical Laboratory
4
Qingdao Blood Center, Qingdao, China
Department of Clinical Laboratory

Abstract
Objectives: To investigate the influence of the anti-tumor necrosis factor-α infliximab (IFX) in the case of rats with silicosis.
Material and Methods: Forty-eight Wistar rats were randomly divided into 3 groups. The study group (N = 16) – silicosis
was induced by intratracheal instillation of  50  mg  silica on day  1, and  IFX was subcutaneously administered at a  dose
of 15 mg/kg of body weight from day 2 to day 6, the vehicle group (N = 16) – silica used as the study group but without IFX,
the sham group (N = 16) – 1 ml of saline was intratracheal-used. Eight rats in each group were euthanized on day 7 and on
day 14, respectively. Lung tissue sections were stained with hematoxylin and eosin or Masson’s trichromedye. The nuclear
factor-κB p65 (NF-κB p65) positioning in the lung tissues were determined by immunohistochemical staining. Levels of tu-
mor necrosis factor α (TNF-α) in rat serum and bronchoalveolar lavage fluid were measured with enzyme linked immuno-
sorbent assay. The inducible nitric oxide synthase (iNOS) mRNA in the lung tissues was measured by quantitative real-time
polymerase chain reaction, as well as inhibitor protein-κB (I-κB) and NF-κB p65 expression were measured quantitatively
by western blotting. Results: Silica installation increased the lung tissues inflammation reaction, oxidative stress and pul-
monary fibrosis. Infliximab treatment significantly improved silica-induced lung pathological changes (inflammatory cells,
collagen deposition), decreased the TNF-α inhibited NF-κB signaling (I-κB, NF-κB p65) as well as oxidant status (iNOS).
Conclusions: Infliximab may improve silica-induced pulmonary inflammation by decreasing the TNF-α, inhibiting NF-κB
signaling (I-κB, NF-κB p65) as well as oxidant status (iNOS), which suggest that IFX has potential role in the treatment of
silica-induced lung damage. Int J Occup Med Environ Health 2018;31(4)

Key words:
Infliximab, Silicosis, TNF-α, Rat model, NF-κB, iNOS

Funding: this work was supported by Qingdao Science and Technology Development (project No. KJZD-12-23-nsh entitled “Effects of Tumor Necrosis Factor-alpha
in Pathogenesis and Therapy of silica Exposed animal Model,” project manager: Hua Zhang, Ph.D.).
Received: June 12, 2016. Accepted: September 25, 2017.
Corresponding author: J. Guo, Qingdao Central Hospital, Department of Clinical Laboratory, 127  Siliu Nan Road, Qingdao  260042, China (e-mail:  Wudao-
jguo2015@126.com).

Nofer Institute of Occupational Medicine, Łódź, Poland 1

 ORIGINAL PAPER     H. ZHANG ET AL.
INTRODUCTION
Silicosis caused by inhalation of crystalline silica is an ir­
reversible potentially fatal lung pneumoconiosis, charac-
terized by inflammation and fibrosis of the lung [1,2]. De-
spite significant progress in its research, the exact mecha-
nism of silicosis has not yet been elicited. Silicosis is be-
lieved to be initiated by an increased number of activated
alveolar macrophages, releasing numerous inflammatory
mediators, which play crucial roles in the occurrence and
development of the disease [3]. Despite extensive effort,
no significant effective treatment method is available to
reverse or slow the disease process [4–6]. Novel approach-
es for key cytokines targeted at treatment of silica-induced
inflammation and fibrosis are urgently needed.
Tumor necrosis factor  α  (TNF-α) as an activator, plays
a  key role in the inflammatory and fibrogenic responses
of silicosis [7]. Exogenous recombinant TNF-α augments
silicotic fibrosis and anti-TNF antibody attenuates silico-
sis, and silicosis are absent in TNF-α receptor knockout
mice [8–10]. Tumor necrosis factor α in a canonical man-
ner triggers nuclear factor-κB  (NF-κB) activation which
may occur in several pathways [11–13]. Nuclear factor-κB
as a transcription factor plays a fundamental role in the
pathogenesis of silicosis. Binding of  NF-κB to  DNA
promotes up-regulation of inducible genes involved in
mediating the inflammatory and fibrotic responses to
silica  [14–16]. Tumor necrosis factor  α also activates in-
ducible nitric oxide synthase  (iNOS) to produce nitric
oxide (NO) in respiratory epithelium [17]. As a reactive
nitrogen species,  iNOS-derived  NO contributes to the
pathogenesis of silica-induced lung disease, up-regulating
inflammatory cytokines and amplifies the inflammatory
response [18,19].
Infliximab (IFX) is a chimeric monoclonal immunoglobu-
lin G1, neutralizing TNF-α biologic activity with a high af-
finity and specificity to human membrane-bound and solu-
ble TNF-α [20–22]. Infliximab has demonstrated a protec-
tive effect on acute lung injury induced by intestinal isch-
2 IJOMEH 2018;31(4)
emia/reperfusion, airway smooth muscle hyper-reactivity
of asthma and cigarette smoking-induced emphysema by
reducing lung inflammation and improving pathological
changes [23–26]. It is also effective in attenuating bleomy-
cin-induced  (BLC-induced) pulmonary fibrosis and  IFX
per se does not result in pulmonary fibrosis [27].
Since no more biochemical and histopathological changes
on pulmonary fibrosis induced by silicosis in the case of rats
by  IFX pretreatment had been reported, the aim of this
study was to examine the protective effects and mechanism
of action of subcutaneously-administered IFX on the silica-
induced lung inflammation and fibrosis in the rat model.
MATERIAL AND METHODS
Dust sampling and suspension preparation
Silica content of the quartz dust (Sigma-Aldrich, St  Lou-
is, MO, USA) was > 99% and the particle size of the dust
was 0.5–10 μm with 80% of the dust particles were 1–5 μm.
Crystalline silica particles were suspended in saline at
50 mg/ml after sterilization (121.3°C, 103.4 kPa for 90 min),
and then added with penicillium to make the final con­
centration of 8000 U/ml in silicon suspension [28].
Animals and experimental design
Male Wistar rats (6–8-week old, 180–220 g in weight) were
obtained from the Food and Drug Administration, Qing­
dao, China and kept on an alternating 12 h light / 12 h dark
cycle under standard temperature (22±2°C) and humid-
ity (60±5%). The rats were acclimatized for 7 days before
the start of the experiment with standard chew food and
water freely available. The animal experimental protocol
complied with the Animal Management Rules of the Chi-
nese Ministry of Health (document No. 55, 2001) and was
approved by the Animal Care Committee, Qingdao Food
and Drug Administration.
Forty-eight rats were randomly divided into 3 experimental
groups according to their experimental treatment as follows:
the treatment group (N = 16), the vehicle group (N = 16)

and the sham group (N = 16). The vehicle group was anes-
thetized with  40  mg/kg pentobarbital sodium via intra-
peritoneal injection and received a  single trans-oral  (TO)
intratracheal instillation of silica suspension of 1 ml as
described previously  [29]. The sham group received the
same procedure but received an equal volume of saline in-
stead of silica. The treatment group received a single dose
of 15 mg/kg IFX (Remicade, Cilag AG Pharmaceutical Com-
pany, Switzerland, registration certificate No. for the import-
ed drug: s20120012) dissolved in 0.2 ml of a diluent consisting
of normal saline subcutaneously on day 2 to 6 for 5 days over-
all after silica installation as described previously [30]. Ani-
mals were sacrificed using sodium pentobarbital (120 mg/rat)
on days 7 or 14 after silica instillation.
Peripheral blood
and bronchoalveolar lavage fluid (BALF)
Blood samples were harvested from abdominal aorta.
Serum was separated by centrifugation and then stored
at –80°C for analyses.
After the blood had been sampled, the right lung was
ligated at the bronchi with surgical wire and  BALF was
collected by washing the left lung with 3 separate aliquots
of 2 ml saline at 37°C through a tracheal cannula. Broncho­
alveolar lavage fluid was centrifuged and stored at –80°C.
Pathologic examination
of lung inflammation and fibrosis
The intact right lung lobes were removed, blotted dry,
the middle lobe was fixed in 10% formalin and processed
for routine histology in paraffin wax. Sections were pre-
pared of 4 mm in size, stained with hematoxylin and eo-
sin (H&E) for histopathology and Masson’s trichrome for
collagen deposition. The lung tissues were examined in
random and blind conditions with standard light micros­
copy. The degree of lung tissue inflammation was evalu-
ated quantitatively by  H&E staining according to its se-
verity. A score ranging from 0 to 4 was designed and the
INTERVENTION OF PNEUMOCONIOSIS     ORIGINAL PAPER
average score was calculated for each group  [15]. The
Masson’s trichrome staining method was used for deter-
mining the severity of fibrosis and the quantitative grade
of pulmonary fibrosis was blindly scored by randomly se-
lecting regions according to the method introduced by Or-
tiz et al. [16]. A score ranging from 0 to 8 was assigned and
the scores were averaged per group.
Immunohistochemical assessment
of nuclear factor-κB p65 (NF‑κB p65) in lung tissue
Lung sections, 4 μm thick, were deparaffinized and then
soaked in hydrogen peroxide  (3%) for  15  min to inacti-
vate endogenous peroxidase antigens. Antigen retrieval
was performed by means of a pressure cooker (ethylene
diaminetetraacetic acid, pH  =  9; for  15  min at  90°C).
Following serum sealing for  30  min, primary anti-
NF‑κB  p65  (Proteintech group,  Inc.,  USA) was added
and incubated for  1  h  at  37°C. The slides were washed
with phosphate buffered saline (PBS) and then incubated
with peroxidase-conjugated anti-rabbit  IgG (Proteintech
group, Inc., USA) for 0.5 h at 37°C. Staining was then per-
formed using a 3,3’-diaminobenzidine staining kit (ZSGB-
Bio Co. Ltd., China). Phosphate buffered saline was used
in the negative control group instead of primary antibody.
A positive reaction was indicated by brown staining in the
nuclei and cytoplasm. The immunohistochemical staining
was evaluated under an Olympus light microscope, Image-
Pro Plus software v. 6.0 (Media Cybernetics, Inc., USA).
Determine TNF-α level in serum and BALF
Tumor necrosis factor α was measured in BALF and se-
rum using commercial enzyme-linked immunosorbent
assay (ELISA) kits (R&D Systems, USA) used according
to the manufacturer’s instructions.
Determine iNOS mRNA
The quantitative analysis of mRNA for iNOS and β-actin
was performed by real-time polymerase chain reaction
IJOMEH 2018;31(4) 3
 ORIGINAL PAPER     H. ZHANG ET AL.
(PCR) using CFX96 real-time PCR detective system (Bio-
Rad Laboratories, Inc., USA) and SYBR Green technol-
ogy. Total RNA was extracted from lungs with the RNA­
isoPlus Kit (Takara Bio, Inc., Japan) according to the man-
ufacturer’s instructions. Ribonucleic acid purity and con-
centration were determined using a  spectrophotometer
NanoDrop2000c (Thermo Fisher Scientific  Inc.,  USA).
Complementary DNA was synthesized from deoxyribonu-
clease-treated total RNA (1 μg) with the PrimeScript RT
reagent kit with gDNA Eraser (Takara Bio, Inc., Japan)
and reverse transcription-real-time  PCR was performed
with the SYBR Premix EX Taq II (Takara Bio, Inc., Japan)
according to the manufacturer’s protocol.
The primers (SangonBioteck,  Co.,  Ltd, China) had the
following sequences:
1. iNOS – forward 5’-ACGGAGAACAGCAGAGTTGG-3’
and reverse 5’-TGTTGGGCTGGGAATAGCAC-3’;
2. β-actin – forward  5’-CCCATCTATGAGGGTTAC-3’
and reverse 5’-TTTAATGTCACGCACGATTTC-3’.
The following thermocycling conditions were used:
initial polymerase activation step for  30  s at  95°C, fol-
lowed by  40  cycles of  5  s  at  95°C  for template denatur-
ation, 30 s at 60°C for annealing and extension. All sam-
ples were amplified by fluorescence measurement in trip-
licates and the mean was used for quantitative real-time
polymerase chain reaction  (RT-qPCR) analysis. Relative
levels of target  mRNA expression were calculated using
the 2–ΔΔCT method and normalized by endogenous control
(β-actin).
Analysis of NF-κB p65 and inhibitor protein-κB (I-κB)
Total protein was extracted from lung tissue using pro-
tein extraction reagents (Shanghai Solarbio Bioscience
& Technology Company, China), and protein concentra-
tions measured using a BCA protein assay kit (Solarbio,
China) according to the manufacturer’s instructions.
Protein extracts were loaded onto a  12%  sodium do-
decyl sulphate- polyacrylamide gel  (SDS-polyacrylamide
4 IJOMEH 2018;31(4)
gel)  (40  μg/well) and electrophoretically transferred to
polyvinylidenedifluoride  (PVDF) membranes. After
blocking for 3 h with 5% skim milk in phosphate buffered
saline with tween (PBST) (PBS with 1% polyoxyethylene-
sorbitan fatty acid esters) buffer at room temperature,
membranes were incubated overnight at  4°C  with poly-
clonal rabbit anti-rat  NF-κB  p65 (1:5000) (Proteintech
Group,  USA), polyclonal rabbit anti-rat I-κB (1:1000)
(Immunoway Inc., USA) in skim-milk PBST. The PVDF
membranes were washed for 3 times with PBST and in-
cubated with peroxidase-conjugated polyclonal goat
anti-rabbit secondary antibody (1:5000) (Beijing Biosyn-
thesis Biotechnology  Co.,  Ltd., China) for  1  h  at room
temperature.
Following final washes, protein signals were visualized
using the enhanced chemiluminescence reagents (Bei-
jing ComWin Biotech Co., Ltd, China) and detected by
the fusion imaging system (Vilber Lourmat Company,
France). Beta-actin protein was visualized and detected
in the same way. The intensity of each signal spot was
quantified using the ImageJ  2x  software (Wayne Ras-
band, National Institutes of Health, USA) that was used
for semiquantifing mean optical density of  NF-κB p65
and I-κB expression.
Statistical analysis
All data is presented as mean (M) ± standard error (SE).
The statistical analyses were performed using  SPSS  22.0
software (SPSS Inc., USA) and GraphPad Prism 6.0 (Graph-
Pad, USA). Statistical significance was determined by one-
way analysis of variance (ANOVA) followed by the post hoc
least significant difference test (LSD-t) and p < 0.05 was
considered statistically significant.
RESULTS
Histopathological findings
Representative sections of rat lung tissue are shown in the
Figure 1. Normal alveolar structure, lack of inflammatory
 INTERVENTION OF PNEUMOCONIOSIS     ORIGINAL PAPER

a) b) c)

d) e) f) Group:
5 control

Pulmonary inflammation [pts]

vehicle
4 treatment
* *
3
2
1
0
7 14
Time after silica instillation [days]
g) h) i)

j) k) l) Group:
5 control
Pulmonary fibrosis [pts]

vehicle
4 treatment *
*
3
2
1
0
7 14
Time after silica instillation [days]

Hematoxylin and eosin (H&E) staining: a) sham group, day 7; b) vehicle group, day 7; c) treatment group, day 7; d) vehicle group, day 14;
e) treatment group, day 14.
Pulmonary inflammation and fibrosis score (0 – normal lung, 8 – total fibrous obliteration of the microscope field): f) on day 7 after instillation
of silica or saline control, l) on day 14 after instillation of silica or saline control. Data presented as mean ± standard error.
* p < 0.05 vehicle group vs. sham group (one-way analysis of variance and least significant difference test).
Masson’s trichrome staining: g) sham group, day 7; h) vehicle group, day 7; i) treatment group, day 7; j) vehicle group, day 14; k) treatment group,
day 14.
Treatment group (N = 16) – silicosis was induced by intratracheal instillation of 50 mg silica on day 1, and IFX was subcutaneously administered
at a dose of 15 mg/kg of body weight from day 2 to day 6.
Vehicle group (N = 16) – received silica as treatment group but without IFX.
Sham group (N = 16) – received the same procedure as vehicle group but with equal volume of saline instead of silica.
Eight rats in each group were euthanized on day 7 and the next 8 rats on day 14 after silica instillation.

Fig. 1. Representative sections (4-μm thickness) of rat lung tissue stained with H&E for histopathology and Masson’s trichrome
for collagen deposition (original magnification ×200)

IJOMEH 2018;31(4) 5
 ORIGINAL PAPER     H. ZHANG ET AL.

cellular infiltration and fibrous thickening, was observed
in the case of the sham rats (Figure 1a).
On day 7 after instillation, acute inflammation and alveo-
lar wall thickening with infiltration of macrophages, lym-
phocytes and neutrophils into the alveoli were observed in
lung tissue of the vehicle group (Figure 1b). On the con-
trary,  IFX  treatment provided protection against silica-
induced lung tissue damage with the reduction of alveoli-
tis severity on day 7 (Figure 1c).
On day 14, animals in the untreated vehicle group showed
extensive pulmonary lesions including multifocal areas of
intense fibrosis with the number of neutrophils decreasing
and the number of lymphocytes increasing. What’s more,
granulomas containing macrophages, neutrophils and fibro-
blasts apparently increased and enlarged, as compared with
day 7 (Figure 1d and 1e). In Masson’s trichrome stained sec-
tions, the sham group had normal collagen fibers distribution
(Figure 1g). In contrast, the untreated vehicle group showed
weak collagen deposition on day 7 (Figure 1h and 1i). In-
creases in collagen deposition began remarkable following
instillation on day  14  (Figure  1j). The degree of fibrosis
was suppressed in the lungs of the silica-administered rats
with IFX treatment (Figure 1k). Inflammation (day 7) and
fibrosis (day 14) score were decreased significantly in the
IFX-treatment group as compared to the vehicle group
(p < 0.05) (Figure 1f and 1l).
Immunohistochemical findings
Sections as shown in the Figure  2a, the sham rats lung
tissue section showed a faint degree of immune staining
for  NF-κB  p65. Silica exposure significantly increased
the NF-κB p65 levels of nuclear protein in the lung tissues,
which was evident from intense brown staining distributed
in nucleus and cytoplasm of alveolar macrophages and

a) b) c)

d) e) f) Group:
0.4 control
NF-κB p65 optical density

vehicle
treatment *
0.3 * *
0.2
0.1
0
7 14
Time after silica instillation [days]

a) section of lung in sham group showing normal architecture, day 7; b) and c) section of lung in vehicle group showing extensive NF-κB p65
expression (brown color) in tissue, day 7 and day 14, respectively; d) and e) in contrast, section of lung in infliximab (IFX) treated group showing
limited NF-κB p65 expression, day 7 and day 14, respectively; f) mean optical density of NF-κB p65 staining in immunohistochemical sections
represents quantification of NF-κB p65 expression (data presented as mean ± standard error).
* p < 0.05, compared with sham/vehicle group (one-way analysis of variance and least significant difference test).
Groups as in Figure 1.

Fig. 2. Immunohistochemical analysis of nuclear factor-κB p65 (NF-κB p65) in rats’ lung sections (original magnification ×400)

6 IJOMEH 2018;31(4)
 INTERVENTION OF PNEUMOCONIOSIS     ORIGINAL PAPER

Table 1. Serum and bronchoalveolar lavage fluid (BALF) levels of tumor necrosis factor α (TNF-α) in rats measured
using enzyme-linked immunosorbent assay (ELISA)

TNF-α concentration
[ng/l]
(M±SE)
Sample
on day 7 on day 14
sham group vehicle group treatment group sham group vehicle group treatment group
(N = 8) (N = 8) (N = 8) (N = 8) (N = 8) (N = 8)
Serum 42.97±4.38 86.41±7.25 66.57±7.02* 45.58±5.16 77.06±4.18 58.73±5.78*
BALF 36.46±4.40 69.46±8.34 49.13±6.09* 38.55±4.57 50.76±5.86 47.35±4.59

M – mean; SE – standard error.

* p < 0.05, compared with sham/vehicle group (one-way analysis of variance and least significant difference test).
Groups as in Figure 1.

epithelial cells (Figure  2b  and  2c). However, treatment
with  IFX markedly inhibited the expression of  NF-κB
p65 (Figure 2d and 2e), as compared to the vehicle group
(Figure 2f) (p < 0.05).
ELISA to determine cytokine levels
in serum and BALF
Serum and BALF levels of TNF-α were shown in the Ta-
ble 1. Serum TNF-α level was found considerably higher
in the vehicle group than in the sham group at both times.
Infliximab treatment significantly decreased TNF-α level
as compared with the vehicle group (Figure 3a) (p < 0.05).
However, upon  IFX treatment,  BALF  TNF-α level was
down-regulated and found only on day 7 (p < 0.05) but
not on day 14 (Figure 3b).
Quantitative real-time PCR analysis
of iNOS mRNA expression
Lung tissues-based iNOS gene expression was shown in
the Table  2. Inducible nitric oxide synthase  mRNA ex-

a) b)
100 100
TNF-α concentration [ng/l]

TNF-α concentration [ng/l]

Group: Group:
sham sham
80 vehicle 80 vehicle
treatment treatment
60 60

40 40

20 20

0 0
7 14 7 14
Time after silica instillation [days] Time after silica instillation [days]

Data presented as mean ± standard error.

* p < 0.05, compared with sham/vehicle group (one-way analysis of variance and least significant difference test).
Groups as in Figure 1.

Fig 3. Tumor necrosis factor α (TNF-α) concentration measured using enzyme linked immunosorbent assay (ELISA) in rats’:
a) serum, b) bronchoalveolar lavage fluid (BALF)

IJOMEH 2018;31(4) 7
 ORIGINAL PAPER     H. ZHANG ET AL.

Table 2. Inducible nitric oxide synthase (iNOS) mRNA expression level in lung tissues of rats measured
using real-time quantitative polymerase chain reaction (RT-qPCR)

iNOS mRNA expression level

(M±SE)
Sample on day 7 on day 14
sham group vehicle group treatment group sham group vehicle group treatment group
(N = 8) (N = 8) (N = 8) (N = 8) (N = 8) (N = 8)
Lung tissue 10.29±0.19 12.91±0.21 11.66±0.14* 10.31±0.16 12.60±0.18 12.03±0.22*

M – mean; SE – standard error.

* p < 0.05, compared with sham/vehicle group (one-way analysis of variance and least significant difference test).
Groups as in Figure 1.

15 duced rats, as compared with the sham group. Treatment

iNOS mRNA expression

Group:
sham
vehicle
treatment
with IFX significantly increased the I-κB protein level on
10 day  7  and  14  (Figure  5b) (p  <  0.05). In contrast to the
downtrend of I-κB levels, relative NF-κB p65 levels (ra-
5 tios of NF-κB p65 to β-actin) were increased in the case
of the silica rats to a larger extent than in the case of the
0 sham animals  (p  <  0.05). However, the increasing was
7 14
Time after silica instillation [days] significantly attenuated in the case of IFX-treatment rats
on day 7 (p < 0.05) but not on day 14 (Figure 5d).
Data presented as mean ± standard error.
* p < 0.05, compared with sham/vehicle group (one-way analysis
of variance and least significant difference test). DISCUSSION
Groups as in Figure 1.
Silicosis is associated with persistent inflammation which
Fig. 4. Inducible nitric oxide synthase (iNOS) mRNA leads to fibroblast formation and excessive collagen de-
expression level in lung tissues of rats measured using real-time
position resulting in interstitial fibrosis [31]. In this study,
quantitative polymerase chain reaction (RT-qPCR)
histopathological analyses have revealed that a  single-
pression level was significantly higher in the vehicle group dose intratracheal silica instillation could produce early
rats than in the sham group, IFX treatment significantly acute alveolitis, characterized by inflammatory cells in-
reduced iNOS mRNA expression on day 7 and 14 as com- filtration into the alveoli, accompanied by early alveolar
pared to the vehicle group (Figure 4) (p < 0.05). walls thickening. Furthermore, granulomas apparently
formed and extensive collagen deposition increased over
Western blot analysis time indicated progressive pulmonary inflammation and
of NF-κB p65 and I-κB protein expression pulmonary fibrosis as compared with the sham group,
The Western blot analysis was employed to semi-quan- closely mimicking acute silicosis injury  [30]. Infliximab-
titatively determine protein expression levels of  NF-κB attenuated silica induced the early inflammation and the
p65 (65 kDa), I-κB (39 kDa), and β-actin (43 kDa) (Fig- subsequent fibrosis, supported by a  marked decrease in
ure 5). Relative I-κB level (ratios of I-κB to β-actin) ana- the distribution and severity of infiltrates, organized gran-
lyzed by densitometry was lower in the case of silica-in- ulomas and collagen deposition following  IFX adminis­

8 IJOMEH 2018;31(4)
 INTERVENTION OF PNEUMOCONIOSIS     ORIGINAL PAPER

a) tration. This result is consistent with previous studies

39 kDa I-κB that indicated the ability of IFX to decrease collagen lev-
els in lung diseases [23–27].
43 kDa β-actin
To explore the possible mechanism(s) of the protective
1 2 3 4 5 6 action of IFX against silica-induced fibrosis, factors of in-
b)
1.5 flammatory, nuclear transcriptional and oxidative stress
I-κB expression [%]

Group:
sham
vehicle were evaluated in the lung tissue.
treatment
1.0 The cytokine TNF-α was believed to be an upstream fac-
tor, which could initiate a cascade complex of inflammato-
0.5 ry mediators in the early inflammatory event and develop
silica-induced fibrosis. Exogenous recombinant  TNF-α
0 augments silicotic fibrosis and anti-TNF antibody attenu-
7 14
Time after silica instillation [days]
ates silicosis, silicosis is absent in the case of TNF-α re-
c) ceptor knockout mice, suggesting that  TNF-α  receptor
signaling is needed for the development of pulmonary
65 kDa NF-κB p65
inflammation and fibrosis [8–10]. In confirmation of the
43 kDa β-actin previous study, we observed that TNF-α level in serum
1 2 3 4 5 6 over the time and in BALF on day 7 increased in the ve-
1.5
d) hicle group to an extent greater than in the sham group,
NF-κB p65 expression [%]

Group:
sham while IFX treatment significantly decreased TNF-α lev-
vehicle
treatment els, which is similar to the trend of histopathological re-
1.0
sults. However, BALF TNF-α level on day 14 didn’t show
the remarkable decreasing upon IFX treatment. We ana-
0.5
lyzed the IFX administration of intraperitoneal injection
which might result in the more unstable drug concentra-
0
7 14 tion distribution in local lung tissues, as compared with
Time after silica instillation [days]
the in-the-blood system.
Nuclear factor-κB has been considered a primary target to
a) a representative western blot.
c) the analysis of the ratio between I-κB and β-actin
accelerate silica-induced inflammation and fibrosis in the
and between NF-κB p65 and β-actin by densitometry. lung [32]. Tumor necrosis factor α induces NF-κB transac-
1 – vehicle group, day 7; 2 – treatment group, day 7; 3 – sham group, tivation by a mechanism involving I-κB degradation [33].
day 7; 4 – vehicle group, day 14; 5 – treatment group, day 14;
6 – sham group, day 14. Tumor necrosis factor-persistent stimulation may lead
Data presented as mean ± standard error. new I-κB–NF-κB complexes to enter the feedback loop,
* p < 0.05, compared with sham/vehicle group (one-way analysis beginning with phosphorylation of I-κB [34]. A pharma-
of variance and least significant difference test).
Groups as in Figure 1. cologic inhibitor  (BAY  11-7085) of  I-κBα  (NF-κBIA)
phosphorylation, inhibiting systemically NF-κB activation,
Fig. 5. Effect of different treatments in the rats’ lung tissues
on the expression of: a) and b) inhibitor protein-κB (I-κB), significantly decreases silica-induced inflammation and fi-
c) and d) nuclear factor-κB p65 (NF-κB p65) brosis by reducing apoptosis and collagen deposition [35].

IJOMEH 2018;31(4) 9
 ORIGINAL PAPER     H. ZHANG ET AL.
In the current study, silica installation induced the ac-
tivation of  NF-κB signaling pathway, which was con-
firmed as followed. Firstly, by immunohistochemical
staining of the lung tissue, silica exposure significantly
increased the  NF-κB  p65 level of nuclear protein dis-
tributed in nucleus and cytoplasm of alveolar macro-
phages and epithelial cells. Secondly, by protein band
analysis of western blotting, silica exposure apparently
increased I-κB degradation and NF-κB p65 expression.
Infliximab administration considerably inhibited  NF-
κB signaling activation by decreasing protein level and
nuclear translocation of NF-κB p65, I-κB phosphoryla-
tion as well. However, the result of NF-κB p65 expres-
sion by  IHC staining kept inconsistency with that by
the  WB analysis on day  14. Considering the method-
ological difference between the 2 technologies, compa-
nied with possibly weakened NF-κB signaling feedback
loop over time, IHC staining result, mainly measuring
combining and dissociative  NF-κB  p65 with intracyto-
plasmic  I-κB and nucleous  DNA, appeared more re-
markable than that by the western blot (WB), which is
measuring merely dissociative ones.
Silica-induced inflammation and oxidative stress envi-
ronment contributes to lung injury. It has been demon-
strated that iNOS (iNOS)-dependent formation of NO
has a  key role in the initiation of pulmonary inflam-
mation and fibrosis  [36]. Quartz instillation into rat
lungs resulted in increasing of mRNA for  iNOS in al-
veolar macrophages [37]. Tumor necrosis factor α may
activate  iNOS to produce  NO in respiratory epithe-
lium [17], which has been reported in alveolar macro-
phages and neutrophils [36,38].
The over-produced iNOS-dependent formation of NO
may cause serious lung damage though the formation of
peroxynitrite (reactive nitrogen species – RNS) combin-
ing with superoxide  [36,39,40]. Furthermore,  NO  pro-
duces and amplifies the inflammatory response by
up-regulating the key cytokine  TNF-α during inflam-
10 IJOMEH 2018;31(4)
mation  [22]. In our study, silica administration in-
creased iNOS expression, which was shown by the quan-
titative real-time PCR analysis of iNOS mRNA expres-
sion, basically identifying with the previous conclusions.
However,  IFX  treatment decreased  INOS expression
on both day  7  and  14. We have supposed that it may
be one of the mechanisms, due to which IFX improves
inflammation and fibrosis by decreasing transcriptional
activation of oxidant-stress index iNOS.
In clinical studies, it has been shown that IFX diminishes
pulmonary fibrosis via blocking  TNF-α  [41,42], and pul-
monary fibrosis is not a  contraindication for  TNF-α in-
hibitor therapy of rheumatology [43]. Altintas et al. [27]
have evaluated the preventive effect of IFX in bleomycin-
induced pulmonary fibrosis, eliminating the harmful effect
of IFX per se on pulmonary fibrosis. However, there is no
study to explain the treatment effect of IFX on silica-in-
duced pulmonary inflammation and fibrosis.
This study has shown that IFX treatment prevents the
inflammatory and fibrotic response as assessed from
quantitative histopathological evaluation of silica-
induced lung fibrosis model in the case of rats. In ad-
dition, it has also been found that  IFX attenuates the
silica-induced inflammatory by decreasing  TNF-α lev-
el, I-κB degradation, NF-κB p65 expression and nuclear
translocation, oxidative stress relative enzyme iNOS ac-
tivities in the damaged lung tissue of rats. To the best
of our knowledge, this is the first study that evaluates
the treatment effect of  IFX on silica-induced lung fi-
brosis in terms of the animal model, but it needs more
indicators and data of pulmonary fibrosis to confirm.
This study hopes to provide drug treatment for pneu-
moconiosis patients to alleviate the pain and improve
the quality of life, so as to provide some help for clinical
diagnosis and treatment. With the Chinese government
to further strengthen the occupational precautionary
measures, the occurrence of occupational diseases will
be significantly reduced.

CONCLUSIONS
In summary, IFX may improve silica-induced pulmonary
inflammation by decreasing the TNF-α, inhibiting NF-κB
signaling (I-κB, NF-κB p65) as well as oxidant status (iNOS),
which suggest that IFX has potential role in the treatment
of silica-induced lung damage.
ACKNOWLEDGMENTS
The authors would like to acknowledge with deep apprecia-
tion and gratitude to the invaluable help of the following per-
sons: You-xin  Ji, M.D.,  Ph.D (North Shore University Hospi-
tal, Manhasset, NY) for this article text corrections.
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